PROJECT SUMMARY Infectious disease is a major threat to human health worldwide. The emergence of antibiotic resistance pathogens necessitates the development of new drugs to treat infection. A fundamental challenge in developing antibiotics is that many pathogens replicate inside host cells rendering them inaccessible to antimicrobial agents. Critical processes for pathogen growth in host cells represent the most promising new targets for therapeutic intervention. Thus, a systematic strategy to identify virulence mechanisms central to establishing growth within the host cell and avoiding killing by host defenses is paramount for defining new targets for therapeutic intervention. Through specialized secretion systems, many intracellular pathogens deploy an arsenal of proteins termed effectors that modulate numerous host cell processes to establish growth. Loss of function of the secretion machinery restricts pathogen survival and replication demonstrating a critical role for effectors in disease. Despite the importance of effectors, very little is known about the events that determine when individual effectors are deployed or their individual contributions during infection. This can largely be attributed to the inability to monitor effector populations in host cells due to low endogenous levels that are undetectable by standard biochemical techniques and redundancy amongst effectors whereby loss of any individual effector does not result in a discernible phenotype. Innovative approaches that overcome the limitations of classic genetic and biochemical approaches are necessary to further our understanding of effector-mediated virulence mechanisms employed by pathogens. BioID is a powerful biochemical technique used to define protein-protein interactions. The goal of the proposed research is to adapt BioID to examine the interactions of bacterial virulence proteins in the context of an infection. Using Legionella pathogenesis as a model system, we will apply this technology to define molecular events central to effector translocation, targeting and function. Our BioID-based experimental system will allow several key questions central to the infection process to be addressed that cannot be resolved using currently available methodologies: 1) What are the host targets of individual effectors? 2) How are effectors distributed once they enter the host cell? 3) When are individual effectors secreted and into which host cell types? 4) What are the signals that trigger effectors selection for secretion? Once established, this technology will be broadly applicable to the study of a variety of pathogenic microorganisms and provide a foundation for defining critical events in disease that is crucial to developing new strategies for therapeutic intervention.